JP2020516865A - Method for simultaneous quantification of ALXN1210 and eculizumab in human serum or urine - Google Patents

Abstract

Methods for simultaneously detecting and quantifying antibodies that bind to the same target and have high sequence identity, present together or alone in a biological sample (eg, eculizumab and ALXN1210) are provided herein. Provided in writing. The method can include analyzing a sample of the proteolytic peptide mixture of the antibodies by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect signature peptides from each antibody in the biological sample. ..

Description

Related Information This application claims benefit of US Provisional Application No. 62/480048, filed March 31, 2017, the priority date of which is hereby incorporated by reference in its entirety. To be done.

BACKGROUND The ability to accurately and reliably determine the pharmacokinetics of a pharmaceutical compound in various preclinical models and samples is essential to the design of clinical trials and dosing regimens to determine optimal efficacy and minimal toxicity. This maximizes the likelihood of success in late-stage clinical trials and is a necessary element in the regulatory approval process. Due to their high success potential and reduced development time and costs, therapeutic monoclonal antibodies (mAbs) are becoming increasingly important to the pharmaceutical industry. These proteins are based on naturally-occurring immunoglobulins (IgG), but modify their natural properties and functions, and bind targets to inhibit their activity or Can be engineered to work by removing Monitoring mAb concentration in serum is generally performed using an enzyme linked immunosorbent assay (ELISA). This technique is sensitive and fast enough to efficiently analyze thousands of samples. However, regarding the quantification of antibodies that bind to the same target or different versions of the same antibody that bind to the same target, it suffers from significant limitations. In addition, immunoassays are susceptible to matrix interference and can have long development times. In addition, the antibody may be rehumanized or otherwise altered between preclinical models, or when transitioning from preclinical samples to clinical samples, or to reduce toxicological or immunological effects in preclinical models and human clinical trials. When re-engineered, the assays often need to be redeveloped as drug development progresses. Therefore, it would be useful to develop other mAb quantification methods that result in improved data quality and reduce development time and cost.

Overview Antibodies that bind to the same target and have high sequence identity, present together or alone in a biological sample (eg, human serum or urine) (eg, eculizumab and ALXN1210 (labrizumab)). Provided herein are methods for simultaneous detection and quantification of This method is important because the two antibodies both bind the same target, but they differ by only 4 amino acids throughout the heavy and light chains.

In one aspect, the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of peptides derived from the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect a signature peptide from each of the antibodies in the biological sample; and (c) internal By quantifying each antibody in the biological sample based on the signal ratio of its signature peptide to a control, wherein the internal control comprises the labeled form of the same signature peptide, to obtain a biological sample ( For example, quantifying and detecting each amount of eculizumab and ALXN1210 present together in human serum or urine). In one embodiment, the eculizumab signature peptide comprises or consists of SEQ ID NO:1 and the ALXN1210 signature peptide comprises or consists of SEQ ID NO:2.

In another aspect, the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide; and (c) based on the signal ratio of the signature peptide to an internal control, By quantifying the amount of eculizumab in the biological sample, where the internal control comprises a labeled form of the same signature peptide, present in the biological sample (eg, human serum or urine) Detecting and quantifying the amount of eculizumab. In one embodiment, the signature peptide comprises or consists of SEQ ID NO:1.

In another aspect, the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide; and (c) based on the signal ratio of the signature peptide to an internal control, By quantifying the amount of ALXN1210 in the biological sample (wherein the internal control comprises a labeled form of the same signature peptide) is present in the biological sample (eg human serum or urine) Detecting and quantifying the amount of ALXN1210. In one embodiment, the signature peptide comprises or consists of SEQ ID NO:2.

In a particular embodiment, a method of detecting and quantifying each amount of two antibodies with high sequence identity present together in a biological sample (eg, human serum or urine), wherein the antibody is eculizumab. And ALXN1210, wherein the method is
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect signature peptides derived from each of the antibodies in the biological sample (wherein eculizumab The signature peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19 and SEQ ID NO: 20, the signature peptide of ALXN1210 is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 22); And (c) quantifying each antibody in the biological sample based on the signal ratio of its signature peptide to an internal control, wherein the internal control comprises a labeled form of the same signature peptide.
A method including is provided.

In another embodiment, a method of detecting and quantifying the amount of antibody present in a biological sample (eg, human serum or urine), wherein the antibody is eculizumab, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) Analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide (wherein the eculizumab signature peptide is SEQ ID NO: 1, SEQ ID NO: 19). And (c) quantifying the amount of eculizumab in the biological sample based on the signal ratio of the signature peptide to an internal control (wherein the internal is selected). Controls contain the same signature peptide in labeled form)
A method including is provided.

In yet another embodiment, a method of detecting and quantifying the amount of antibody present in a biological sample (eg, human serum or urine), wherein the antibody is ALXN1210 and the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) Analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide (wherein the signature peptide of ALXN1210 is SEQ ID NO:2, SEQ ID NO:21). And (c) quantifying the amount of ALXN1210 in the biological sample based on the signal ratio of the signature peptide to an internal control (wherein the internal is selected). Controls contain the same signature peptide in labeled form)
A method including is provided.

The methods described herein may include additional steps. For example, in one embodiment, the method further comprises contacting the biological sample with an affinity capture reagent, such as bead-loaded Protein A, prior to treatment with a protease (eg, trypsin). In another embodiment, the method further comprises washing the Protein A binding antibody to remove unbound components prior to proteolysis. In another embodiment, the method further comprises denaturing the antibody sample. In another embodiment, the method further comprises reducing the antibody sample. In another embodiment, the method further comprises alkylating the antibody sample. In one embodiment, denaturing, reducing and alkylating unfold the antibody protein and facilitate proteolytic digestion.

Any suitable mass spectrometry based assay may be used in the methods described herein. In another embodiment, the mass spectrometry based assay is reverse phase UPLC-MS/MS.

The signature peptides used in the methods described herein do not exceed 20 amino acids in length. For example, in one embodiment the signature peptide does not exceed 20, 19, 18, 17, 16 or 15 amino acids in length. In one embodiment, the signature peptide comprises or consists of SEQ ID NO:1. In another embodiment, the signature peptide comprises or consists of SEQ ID NO:2. In one embodiment, the eculizumab signature peptide is SEQ ID NO:1. In another embodiment, the eculizumab signature peptide is SEQ ID NO:19. In another embodiment, the eculizumab signature peptide is SEQ ID NO:20. In another embodiment, the ALXN1210 signature peptide is SEQ ID NO:2. In an embodiment, the signature peptide of ALXN1210 is SEQ ID NO:21. In yet another embodiment, the ALXN1210 signature peptide is SEQ ID NO:22.

Further provided is an isolated peptide consisting of a particular sequence. In one embodiment, an isolated peptide consisting of SEQ ID NO: 1 is provided. In another embodiment, an isolated peptide consisting of SEQ ID NO:2 is provided. In yet another embodiment, an isolated peptide consisting of SEQ ID NO: 19 is provided. In a further embodiment there is provided an isolated peptide consisting of SEQ ID NO:20. In another embodiment is provided an isolated peptide consisting of SEQ ID NO:21. In another embodiment, an isolated peptide consisting of SEQ ID NO:22 is provided.

FIG. 1 is a schematic diagram of a hybrid immunocapture liquid chromatography mass spectrometry assay.

FIG. 2 shows the heavy and light chain sequences of eculizumab and ALXN1210. The four amino acid differences between the eculizumab and ALXN1210 heavy chain sequences are underlined.

FIG. 3 shows the effect of different detergents used during protease digestion on the final signature peptide signal in mass spectrometry.

Figure 4 shows the effect of detergent concentration on the ratio of analytes measured compared to a labeled internal standard of the same peptide at a known concentration.

Figure 5 shows the effect of 1% DMSO added to the mobile phase of the chromatographic steps of the procedure on the final signal of the signature peptide in mass spectrometry.

FIG. 6 shows a representative extracted ion chromatogram (XIC) of a blank sample.

FIG. 7 shows a representative extracted ion chromatogram (XIC) of the LLOQ sample.

Detailed Description Eculizumab, also known as Soliris®, is a humanized monoclonal antibody produced in a single unit dosage form containing 300 mg (10 mg/ml) in 30 ml of injectable solution. It is a humanized monoclonal antibody with binding specificity for somatic protein C5. The eculizumab V regions and their humanization are described in US Pat. No. 6,355,245, the teachings of which are expressly incorporated herein by reference. Eculizumab is composed of 1324 amino acids with a molecular weight of approximately 148 kDa.

The heavy chain sequences of eculizumab CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 3, 4 and 5, respectively. The light chain sequences of eculizumab CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 6, 7 and 8, respectively. The heavy chain variable region sequence of eculizumab is shown in SEQ ID NO:9 and the light chain variable region sequence is shown in SEQ ID NO:10. The eculizumab heavy and light chain sequences are set forth in SEQ ID NOS: 12 and 13, respectively.

ALXN1210 is an anti-C5 antibody described in International Patent Application No. PCT/US2015/0192225 and US Patent No. 9,079,949, the teachings of which are expressly incorporated herein by reference. is there. ALXN1210 is a humanized monoclonal antibody that is highly structurally related to eculizumab. Recently, ALXN1210 has been given the generic name labrizumab, and it has four unique amino acid substitutions for the purpose of enhancing the duration of terminal complement inhibition while maintaining important eculizumab characteristics such as epitopes and low immunogenicity. Introduced into the chain was generated via minimal target manipulation of eculizumab. Thus, labrizumab (ALXN1210) and eculizumab showed 99%, except that labrizumab has a T1/2 of greater than 40 days in humans compared to eculizumab T1/2 of approximately 12 days in humans. They share greater primary sequence identity and have highly similar pharmacology. Labrizumab and ALXN1210 are used interchangeably herein. ALXN1210 selectively binds to human complement protein C5 and inhibits its cleavage to C5a and C5b during complement activation.

The heavy chain sequences of CDR1, CDR2 and CDR3 of ALXN1210 are shown in SEQ ID NOs: 17, 18 and 5, respectively. The light chain sequences of CDR1, CDR2 and CDR3 of ALXN1210 are set forth in SEQ ID NOs: 6, 7 and 8, respectively. The heavy chain variable region sequence of ALXN1210 is shown in SEQ ID NO:14. The light chain variable region sequence of ALXN1210 is shown in SEQ ID NO:10. The entire heavy chain sequence of ALXN1210 is shown in SEQ ID NO:16. The entire light chain sequence of ALXN1210 is shown in SEQ ID NO:13.

The present disclosure provides for accurate simultaneous quantification of eculizumab and ALXN1210 in biological samples (eg, human serum or urine) from a patient, particularly in clinical situations where the patient switches from one antibody to another during the course of treatment. Address an important need to The methods described herein can be adapted for switching from eculizumab to other anti-C antibodies or other complement inhibitors. Such quantitation is important for determining the precise dosing regimen. Simultaneous quantification of eculizumab and ALXN1210 is very similar because the sequences of these two antibodies are highly similar (only 4 amino acids differ as described) and because they bind the same target. Have difficulty. The methods described herein will be readily adapted by switching from eculizumab to alternative complement inhibitors that have more structural differences with eculizumab than ALXN1210. Ligand binding assays such as ELISA quantitate antibodies by epitope, so the preciseness of eculizumab and ALXN1210 is accurately quantified by their sequence similarity and by the fact that these antibodies both bind human C5 at the same epitope. Is not enough to do. Mass spectrometry relies on unique amino acid (peptide) sequences to distinguish proteins. Therefore, given the greater than 99% sequence identity between eculizumab and ALXN1210, past mass spectrometric assays lacked the sensitivity or selectivity required to distinguish these antibodies. This problem is also due to the fact that human sera share a large number of endogenous antibodies (many of which also share regions of sequence identity and/or homology with eculizumab and ALXN1210, thus creating additional noise and accurate quantification of antibodies. It is exacerbated by the fact that it contains

Experimental data showing that the assay described in the present disclosure solves the above problems and can reliably detect and quantify eculizumab and ALXN1210 in human serum and urine samples using the methods described herein. I will provide a.

Digestion A biological sample containing eculizumab and/or ALXN1210, such as serum or urine, was treated with a protease to produce a signature peptide for analysis by mass spectrometry, regardless of the initial capture step Form a digested antibody sample containing one or more signature peptides of As used herein, the term “biological sample” refers to any component derived from or separated from an animal or human patient, including urine, blood, plasma and serum.

As used herein, the term "signature peptide" refers to a peptide that exhibits experimentally advantageous chromatographic and mass spectrometric properties and that is unique (ie, specific for eculizumab or ALXN1210). Point to. As used herein, "advantageous chromatographic performance" can be defined as narrow peaks, low background noise and high peptide recovery. As used herein, "good mass spectrometric performance" may be indicated by relatively high parent and fragment ionic strengths with a high degree of selectivity for the signature peptide sequence. In one embodiment, the signature peptide does not exceed 20, 19, 18, 17, 16 or 15 amino acids in length. In another embodiment, the eculizumab signature peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:19 and SEQ ID NO:20. In another embodiment, the ALXN1210 signature peptide is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:21 and SEQ ID NO:22.

The term "protease" as used herein refers to an enzyme capable of cleaving or hydrolyzing a peptide or protein into fragments in a specific or general random manner. Examples of proteases include trypsin, papain, endoproteinase LysC, endoproteinase ArgC, staph aureus V8, chymotrypsin, Asp-N, Asn-C, pepsin and endoproteinase GluC.

In one embodiment, the biological sample is first contacted with an affinity capture reagent prior to digestion. After washing with an appropriate biological buffer system, the concentrated biological sample is then eluted and treated with protease. As used herein, the term “affinity capture reagent” refers to an antibody capture reagent or antigen immobilized on a solid substrate. In one embodiment, the affinity capture reagent is protein A or G. In another embodiment, the affinity capture reagent is an antibody or target antigen of complement protein C5 or complement protein C5 attached to a solid substrate. In another embodiment, the affinity capture reagent is biotinylated. In another embodiment, Protein A or G is immobilized or conjugated on a matrix and placed in a chromatography column format. In another embodiment, Protein A or G may be immobilized on magnetic beads, unbound material washed away and attached to the beads while the bound antibody may be digested with a protease.

After contacting and binding the biological sample with the affinity reagent, it is then washed to remove non-specifically bound host proteins or other biomolecules. In certain embodiments, the antibody bound to the affinity capture reagent is denatured to facilitate efficient elution from the affinity reagent and complete protease digestion. In one embodiment, denaturing is performed using a surfactant. In one particular embodiment, RAPIGEST™ is used to perform the denaturation. In another particular embodiment, PROTEASEMAX™ is used to perform the denaturation. In certain embodiments, the antibody bound to the affinity reagent is also reduced to break disulfide bonds and further facilitate protease digestion. In a particular embodiment, the reduction is carried out using dithiothreitol (DTT). In another embodiment, the reducing antibody is alkylated to prevent disulfide bond reformation. In one embodiment, iodoacetic acid is used to carry out the alkylation.

High Performance Liquid Chromatography and Mass Spectrometry After digestion, high performance liquid chromatography in combination with mass spectrometry is used to analyze the treated samples to quantify the amount of each antibody present in the biological sample.

Mass spectrometry techniques are well known in the art (eg reviewed in Yates et al., Annu Rev Biomed Eng. 2009; 11:49-79). In one embodiment, mass spectrometry is performed by liquid chromatography/mass spectrometry (LC/MS). Other forms of mass spectrometry that can be used include, for example, ultra high performance liquid chromatography (UPLC) or tandem mass spectrometry (MS/MS). In a particular embodiment, reverse phase liquid chromatography tandem mass spectrometry (RPLC/MS/MS) is used. Ionization techniques that can be used include electron impact ionization (EI), chemical ionization (CI), desorption chemical ionization (DCI), fast atom bombardment (FAB), atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), Matrix assisted laser desorption/ionization (MALDI) is included. Mass spectrometers that can be used include quadrupole, time of flight (TOF), orbitraps and linear ion traps. In some embodiments, the analyzer is used in a tandem MS. In one embodiment, a triple quadrupole analyzer is used.

As used herein, "chromatography" means that a chemical entity flows around or on a stationary liquid phase or solid phase and is carried by a liquid or gas as a result of the differential distribution and separation of the chemical entity. Refers to the process of separating a chemical mixture into components.

As used herein, "liquid chromatography" (LC) refers to the uniform solution of fluid through a column of defined fine matter (ie, particles) or through capillary passages to determine the By the process of separating components. A differential amount of time is spent in association between one or more stationary phases and the bulk fluid (ie, mobile phase), as the fluid moves relative to the stationary phase, the size of the mixture and/or Alternatively, the separation occurs due to the distribution of components according to the chemical characteristics. Liquid chromatography includes high performance liquid chromatography (HPLC), reverse phase liquid chromatography (RPLC) and high performance turbulent flow liquid chromatography (HTLC), hydrophilic interaction liquid chromatography (HILIC) and normal phase liquid chromatography ( NPLC).

Chromatographic columns typically include a medium (ie, packing material) to facilitate separation (ie, fractionation) of chemical moieties. The medium may include microparticles. The particles may include a binding surface that covalently binds to different chemical functional groups that interact with various chemical moieties within the sample mixture to facilitate separation of the chemical moieties, such as the signature peptides quantified in the experiments herein. Separation often depends on both size and chemical characteristics. One suitable binding surface is a hydrophobic binding surface such as an alkyl binding surface. The alkyl-bonded surface may include C4, C8 or C18-bonded alkyl groups to further separate hydrophobic entities. The chromatography column includes an inlet for receiving the sample and an outlet for discharging the effluent containing the fractionated and separated sample components. The digested antibody sample is applied to the column at the inlet, eluted with the solvent or solvent mixture or solvent gradient and discharged at the outlet. Different solvent modes can be selected to elute different peptides of interest. For example, liquid chromatography can be performed using gradient mode, isocratic mode or polytype (ie mixed) mode. In one embodiment, the starting solvent or sample loading solvent is an aqueous solvent containing 5% organic modifier, such as acetonitrile, with elution occurring as the concentration of acetonitrile increases to 50% over 50 minutes. In some embodiments, additional mobile phase additives are used. In other embodiments, the mobile phase additive is DMSO.

The chromatographically separated components can be combined with a mass spectrometer to measure the molecular weight of the particular fraction present in each peak eluting from the chromatography column. The data from the mass spectrometry can then be used to detect and quantify the amount of the signature peptide as it elutes from the column, thereby art-recognized, as discussed below. Techniques can be used to calculate and compare each amount of antibody present in a biological sample.

Quantitation Quantitation of the signature peptide, normalized to a similar internal standard peptide, can be achieved using calculations from a standard curve.

In one embodiment, the absolute quantification of the signature peptide and the corresponding source antibody is determined. Quantitative determination of signature peptides can be performed using an internal standard peptide, such as a labeled form of the signature peptide (i.e., they are structurally and chemically similar to the signature peptide, but are Is detectably different). Thus, the eluate from the separation process contains an amount of the signature peptide proportional to the known amount of labeled internal standard peptide in the sample. The standard curve then makes it possible to relate the amount of the signature peptide in the sample to the amount of the antibody from which it is derived. In one embodiment, the internal standard peptide is isotopically labeled and incubated with the antibody fragment at a defined concentration. As used herein, an “isotopic label” is one or more heavy atom isotopes (eg, stable isotopes such as deuterium, ¹³ C, ¹⁵ N, ¹⁸ O, ³⁷ Cl or ^81). Br) refers to peptides that are synthetically enriched.

Methods Antibodies that bind to the same target and have high sequence identity, present together or alone in a biological sample (eg, human serum or urine) (eg, eculizumab and ALXN1210 (labrizumab)). Provided herein are methods for simultaneous detection and quantification of

In one aspect, the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of peptides derived from the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect a signature peptide from each of the antibodies in the biological sample; and (c) internal By quantifying each antibody in the biological sample based on the signal ratio of its signature peptide to a control, wherein the internal control comprises the labeled form of the same signature peptide, to obtain a biological sample ( For example, quantifying and detecting each amount of eculizumab and ALXN1210 present together in human serum or urine). In one embodiment, the eculizumab signature peptide comprises or consists of SEQ ID NO:1 and the ALXN1210 signature peptide comprises or consists of SEQ ID NO:2.

In another aspect, the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide; and (c) based on the signal ratio of the signature peptide to an internal control, By quantifying the amount of eculizumab in the biological sample, where the internal control comprises a labeled form of the same signature peptide, present in the biological sample (eg, human serum or urine) Detecting and quantifying the amount of eculizumab. In one embodiment, the signature peptide comprises or consists of SEQ ID NO:1.

In another aspect, the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide; and (c) based on the signal ratio of the signature peptide to an internal control, By quantifying the amount of ALXN1210 in the biological sample (wherein the internal control comprises a labeled form of the same signature peptide) is present in the biological sample (eg human serum or urine) Detecting and quantifying the amount of ALXN1210. In one embodiment, the signature peptide comprises or consists of SEQ ID NO:2.

In one embodiment, a method of detecting and quantifying each amount of two antibodies with high sequence identity present together in a biological sample (eg, human serum or urine), wherein the antibody comprises eculizumab and ALXN1210, and the method is
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect signature peptides derived from each of the antibodies in the biological sample (wherein eculizumab The signature peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19 and SEQ ID NO: 20, the signature peptide of ALXN1210 is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 22); And (c) quantifying each antibody in the biological sample based on the signal ratio of its signature peptide to an internal control, wherein the internal control comprises a labeled form of the same signature peptide.
A method including is provided.

In another embodiment, a method of detecting and quantifying the amount of antibody present in a biological sample (eg, human serum or urine), wherein the antibody is eculizumab, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) Analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide (wherein the eculizumab signature peptide is SEQ ID NO: 1, SEQ ID NO: 19). And (c) quantifying the amount of eculizumab in the biological sample based on the signal ratio of the signature peptide to an internal control (wherein the internal is selected). Controls contain the same signature peptide in labeled form)
A method including is provided.

In yet another embodiment, a method of detecting and quantifying the amount of antibody present in a biological sample (eg, human serum or urine), wherein the antibody is ALXN1210 and the method comprises
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) Analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide (wherein the signature peptide of ALXN1210 is SEQ ID NO:2, SEQ ID NO:21). And (c) quantifying the amount of ALXN1210 in the biological sample based on the signal ratio of the signature peptide to an internal control (wherein the internal is selected). Controls contain the same signature peptide in labeled form)
A method including is provided.

The methods described herein may include additional steps. For example, in one embodiment, the method further comprises contacting the biological sample with an affinity capture reagent, eg, bead-loaded protein A, before treating it with a protease (eg, trypsin). In another embodiment, the method further comprises washing the Protein A binding antibody to remove unbound components prior to proteolysis. In another embodiment, the method further comprises denaturing the antibody sample. In another embodiment, the method further comprises reducing the antibody sample. In another embodiment, the method further comprises alkylating the antibody sample. In one embodiment, denaturing, reducing and alkylating unfold the antibody protein and facilitate proteolytic digestion.

Isolated Peptides Further provided are isolated peptides consisting of a particular sequence. In one embodiment, an isolated peptide consisting of SEQ ID NO: 1 is provided. In another embodiment, an isolated peptide consisting of SEQ ID NO:2 is provided. In yet another embodiment, an isolated peptide consisting of SEQ ID NO: 19 is provided. In a further embodiment there is provided an isolated peptide consisting of SEQ ID NO:20. In another embodiment is provided an isolated peptide consisting of SEQ ID NO:21. In another embodiment, an isolated peptide consisting of SEQ ID NO:22 is provided.

One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be covered by the following claims. In addition, all references cited throughout this application are incorporated herein by reference in their entirety, as if individually incorporated by reference. All sequence listing information is also part of this disclosure.

Example 1: Hybrid immunocapture UPLC-MS/MS approach for simultaneous detection and quantification of ALXN1210 and eculizumab in human serum The following hybrid immunocapture for simultaneous detection and quantification of ALXN1210 and eculizumab in human serum samples. A UPLC-MS/MS approach was developed (schematically shown in Figure 1). Due to the high molecular weight of ALXN1210 and eculizumab, a practical direct quantitative analysis using the LC/triple quadrupole mass spectrometry technique was not feasible. An immunoaffinity capture approach was developed to enrich ALXN1210 and eculizumab from human serum using magnetic beads coated with Protein A. The extracted natural and drug antibody proteins were subjected to on-bead proteolysis: denaturation with low concentrations of organic solvent, reduction with dithiothreitol (DTT) at 60° C., alkylation with iodoacetic acid, and simultaneous with or after the protease trypsin. Digestion. The characteristic "signature" peptides generated from each antibody after trypsin proteolysis were then used as a surrogate for the detection and quantification of ALXN1210 and eculizumab in human serum using UPLC-MS/MS. did. Two signature tryptic peptides were selected for monitoring in the assay, as described below.

Example 2: Identification of ALXN1210 and Eculizumab Signature Peptide Sequences ALXN1210 and eculizumab share significant sequence identity (ie, as shown in Figure 2, four amino acid differences between the two antibody drugs). Exists only). Therefore, for specific detection of each antibody, in silicotrypsin digestion was used to generate a signature peptide unique to each of ALXN1210 and eculizumab. As shown in Table 1, six peptides containing amino acid differences between the sequences of the two antibodies were selected.

Example 3: Immunocapture of ALXN1210 and eculizumab in human serum An immunocapture assay was developed for the enrichment of ALXN1210 and eculizumab from human serum samples as described below. Sensitivity was one of the major challenges in assay development. The presence of large amounts of endogenous protein from other capture antibodies and other components in the biological matrix results in high background, significant ion suppression, and quantitation interference. Protein A was used to test the immunocapture approach. As a control, whole serum direct digestion was used.

Protein A is a protein of microbial origin with the ability to bind human IgG1, IgG2 and IgG4 with high affinity for the antibody Fc region. Even when both eculizumab and ALXN1210 contain non-naturally occurring protein engineered heavy chains constructed with both IgG2 and IgG4 elements, both still retain the ability to bind protein A. During the incubation period, Protein A magnetic beads were incubated with a human serum sample and IgG from the sample was bound to the beads via the Fc region (PUREPROTEOME™ Protein A magnetic beads, EMD Millipore, product number LSKMAGA10). An external magnet (PUREPROTEOME™ magnetic stand, Millipore, product number LSKMAGS15) was used to separate and wash the IgG-Protein A bead complex from other unbound serum components. Protease digestion was performed directly on the bead-bound proteins using MS grade trypsin protease (Pierce product number 90305B).

For whole serum digestion, direct trypsin digestion of samples was performed without any pretreatment to concentrate target protein or reduce background. It was found that the signature peptide was recovered using the whole serum digestion approach. Background was higher than that observed using other immunocapture approaches. However, the analyte response (detection) from the Protein A capture was high, giving excellent sensitivity and quantitation. Even when spiked QCs were examined, this technique using protein A capture showed high accuracy and precision.

Trypsin digestion often used specific detergents to denature proteins and maintain solubility for better digestion and recovery. During method development, RAPIGEST™, a detergent that preferentially unfolds antibodies before proteases, was first evaluated to aid protein digestion. Alternatively, recovery of the signature peptide was not optimal, so another surfactant PROTEASEMAX™ was tested for comparison. As shown in FIG. 3, PROTEASEMAX™ was found to be significantly more effective than RAPIGEST™. Surprisingly, further investigation showed that the digestion efficiency was highest when PROTEASEMAX™ was not added, as shown in FIG. Importantly, these results suggested that denaturation was not necessary for complete digestion and recovery of the antibody, and detergents could adversely affect the enzymatic digestion of certain antibodies.

Example 4: Simultaneous quantification of ALXN1210 and eculizumab using the UPLC-MS/MS approach After immunocapture and protease digestion, a sample of the proteolytic peptide mixture was subjected to reverse phase UPLC-MS/MS. ALXN1210 and eculizumab were used as anti-C5 antibodies with high affinity for complement protein C5. This assay was desirable because clinical trials were in progress to switch one antibody to the other for treatment and required quantification of their relative amounts at any given time during the switching study. . Since the assay was further complicated by varying levels of endogenous C5 bound to the administered antibody, the effect of C5 concentration on the recovery of the signature peptide from ALXN1210 and eculizumab was evaluated. In addition to normal human C5 level sera, two levels of C5 concentration (C5 depletion, endogenous C5 levels in normal human serum, and enriched 100 μg/mL in normal human serum) at three different levels were used. Quality controls (low C5 concentration and high C5 concentration) were prepared. As shown in Tables 2 and 3 below, C5 concentration had no effect on the detection and quantification of ALXN1210 and eculizumab, respectively.

The effect of DMSO as a mobile phase additive was then investigated. The following chromatographic conditions were used (with or without 1% DMSO):
LC pumps: One Agilent 1100 series pumps and Agilent 1200 SL series pumps Pre-column frits: 2-μm stainless steel in-line solvent filters, Upchurch Scientific-Rheodyne, product number A-103x.
Analytical column: Acquity BEH C8, 2.1 mm x 50 mm, 1.7 μm, Waters, Product No. 1 86002877.
Column temperature: 50°C
Pump Program: See Methods Mobile Phase A: 100:0.1, Water/Formic Acid Mobile Phase B: 100:0.1:1, Acetonitrile/Formic Acid/DMSO.
Flow rate: 0.4-0.5 mL/min Injection volume: 25 μL
LC pressure: about 280 bar
Autosampler wash 1:5:25:30:40:0.1 TFE/IPA/ethanol/acetonitrile/formic acid, v/v/v/v/v
Autosampler washing 2:80:20:0.1, water/MeOh/formic acid Approximate execution time: 7.5 minutes

The peptide elution gradient pump program, make-up pump program, ACH valve program, MS conditions and other parameters are shown in Tables 4, 5, 6, 7 and 8, respectively.

As shown in FIG. 5, when 1% DMSO was added to mobile phase B, the response of ALXN1210 signature peptide was doubled (DMSO, Thermo Scientific, product number TS20684). In the case of eculizumab, the original precursor ion ([M+3H]) was completely converted to the precursor ion [M+2H], which was selected for quantification. The sensitivity of eculizumab increased by about 15%. The peptide elution and column wash gradients used are shown in Table 4 below.

Next, carryover from different phase columns was evaluated. In this method, the C18 reverse phase column was initially selected. Considerable carryover was observed due to the relative hydrophobicity of the two signature peptides (about 103% for ALXN1210 and about 118% for eculizumab peptide compared to LLOQ levels in the blank immediately after ULOQ). The C8 column was then evaluated and ALXN1210 and eculizumab peptide carryover were significantly reduced to less than 50% and 30%, respectively, which was acceptable due to injection sequence protection.

Overall, the hybrid immunocapture UPLC-MS/MS method was successfully validated for the detection of eculizumab and ALXN1210 and quantification using the signature trypsin peptides from each (SEQ ID NOS: 1 and 2, respectively). The standard curve range was 1.00-500 μg/mL for ALXN1210 and 5.00-500 μg/mL for eculizumab, using a diluted serum sample volume of 25 μL and an MRD of 10. Using linear regression, a correlation coefficient of >0.996 was observed for both mAbs during validation. As shown in Tables 9 and 10, respectively, for both ALXN1210 and eculizumab, excellent intraday and daily assay precision and accuracy from all quality control (QC) concentration levels were demonstrated. The method was also successfully cross-validated using ALXN1210 and eculizumab individual ligand binding assays.

Example 5: Quantification of ALXN1210 in human urine Matrix effects present in urine samples can prevent mAb quantification using traditional ligand binding assays. In order to overcome these problems, the above UPLC-MS/MS method was optimized to quantify ALXN1210 in human urine. Briefly, ALXN1210 was subjected to denaturation, reduction with dithiothreitol (DTT) at 65°C, alkylation with iodoacetamide, and trypsin digestion. The signature peptide (SEQ ID NO:2) generated from ALXN1210 was then used for quantification of ALXN1210 using UPLC-MS/MS. Tables 11 and 12 list the equipment and reagents used, respectively.

Carryover was one of the major technical challenges during the development of assays for the detection of ALXN1210 and eculizumab in human serum. The HPLC gradient of the human urine assay was improved to include a higher proportion of starting mobile phase B (14% to 25%). This change resulted in no detectable carryover, eliminated additional column wash cycles, and reduced sample run time. Carryover performance during verification is shown in Table 13.

Often, proteins and red blood cells are present in the urine of patients, so further studies were performed to assess the performance of the assay under conditions of high protein and red blood cell (hemolysis) levels. Urine interference quality control (QC) samples containing 15% serum or 2% hemolyzed whole blood (% by volume) were prepared. The standard sample ("std-") did not contain any serum and hemolysis. As shown in Table 14, the recovery of low QC levels of ALXN1210 signature peptide with 15% serum was substantially reduced compared to the QC sample prepared with the calibration curve and 100% urine matrix.

During sample processing, human serum was added to all samples (5% by volume of sample) to normalize the calibration curve QC and QC samples containing serum or hemolyzed whole blood. Standard samples ("std-") and plate acceptance QC samples ("Low-", "Mid-" and "High-") did not contain any serum or hemolyzed blood. The digestion period was then extended to facilitate the recovery of ALXN1210 signature peptide in urine samples containing human serum. As shown in Table 15, at LLOQ QC levels, this condition provided good recovery for both 15% serum and 2% hemolyzed whole blood. The assay also showed good sensitivity with a sample volume of 20 μl. Representative extracted ion chromatograms (XIC) of the blank and LLOQ are shown in FIGS. 6 and 7.

In summary, the UPLC-MS/MS method for the quantification of ALXN1210 in human urine using the signature trypsin peptide was successfully developed and validated. At a sample volume of 20 μL, the calibration curve range of the assay was 0.0800-40.0 μg/mL for ALXN1210. Pooled human urine can be used to dilute samples above ULOQ up to 10-fold and 2-fold (data not shown). For linearity, recovery, dilution integrity, processed sample stability, QC benchtop stability, matrix effect, batch size, reinjection stability, QC freeze/thaw stability, carryover, and interference from IS analytes In addition, it demonstrated excellent intraday and daily assay precision and accuracy from all quality control (QC) concentration levels (data not shown). This method overcomes matrix effects in urine and demonstrates the ability of the UPLC-MS/MS method to quantify ALXN1210 with excellent sensitivity.

Claims (41)

A method of detecting and quantifying each amount of two antibodies having high sequence identity present together in a biological sample, said antibodies being eculizumab and ALXN1210, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect signature peptides derived from each of the antibodies in the biological sample, wherein , The signature peptide of eculizumab comprises SEQ ID NO:1 and the signature peptide of ALXN1210 comprises SEQ ID NO:2; and (c) based on the signal ratio of the signature peptide to the internal control, in the biological sample. Quantifying each antibody, wherein the internal control comprises comprising the same signature peptide in labeled form. A method of detecting and quantifying the amount of antibody present in a biological sample, said antibody being eculizumab, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect a signature peptide, wherein the signature peptide comprises SEQ ID NO:1; And (c) quantifying the amount of eculizumab in the biological sample based on the signal ratio of the signature peptide to the internal control, wherein the internal control determines the labeled form of the same signature peptide. A method, including including. A method of detecting and quantifying the amount of antibody present in a biological sample, said antibody being ALXN1210, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect the signature peptide, wherein the signature peptide comprises SEQ ID NO:2; And (c) quantifying the amount of ALXN1210 in the biological sample based on the signal ratio of the signature peptide to the internal control, wherein the internal control determines the labeled form of the same signature peptide. A method, including including. The method according to any of the preceding claims, wherein the protease is trypsin. The method of any of the preceding claims, further comprising contacting the biological sample with an affinity capture reagent prior to treating the biological sample with a protease. The method of claim 5, wherein the affinity capture reagent is bead-loaded Protein A. 7. The method of claim 5 or 6, further comprising washing the Protein A binding antibody to remove unbound components prior to proteolysis. The method of any of the preceding claims, further comprising denaturing the antibody sample. The method of any of the preceding claims, further comprising reducing the antibody sample. The method according to any of the preceding claims, further comprising alkylating the antibody sample. The method of any of the preceding claims, wherein the denaturing, reducing, and alkylating unfold the antibody protein and facilitate proteolytic digestion. The method according to any of the preceding claims, wherein the mass spectrometry is reverse phase UPLC-MS/MS. The method according to any of the preceding claims, wherein the biological sample is serum or urine. The method according to any of the preceding claims, wherein the signature peptide comprises no more than 20 amino acids. The method according to any of the preceding claims, wherein the signature peptide comprises no more than 15 amino acids. An isolated peptide consisting of SEQ ID NO:1. An isolated peptide consisting of SEQ ID NO:2. A method of detecting and quantifying each amount of two antibodies having high sequence identity present together in a biological sample, said antibodies being eculizumab and ALXN1210, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect signature peptides derived from each of the antibodies in the biological sample, wherein The signature peptide of eculizumab is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19 and SEQ ID NO: 20, and the signature peptide of ALXN1210 is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 22. And (c) quantifying each antibody in the biological sample based on the signal ratio of its signature peptide to an internal control, wherein the internal control has the same signature in labeled form. A method comprising including a peptide. A method of detecting and quantifying the amount of antibody present in a biological sample, said antibody being eculizumab, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect a signature peptide, wherein the signature peptide of eculizumab is SEQ ID NO: 1, Selecting from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20; and (c) quantifying the amount of eculizumab in the biological sample based on the signal ratio of the signature peptide to an internal control. , Wherein the internal control comprises a labeled form of the same signature peptide. A method of detecting and quantifying the amount of antibody present in a biological sample, said antibody being ALXN1210, said method comprising:
(A) treating the biological sample containing the antibody with a protease to form a proteolytic peptide mixture of the antibody in the biological sample;
(B) analyzing a sample of the proteolytic peptide mixture by high performance liquid chromatography (HPLC) tandem mass spectrometry to detect a signature peptide, wherein the signature peptide of ALXN1210 is SEQ ID NO:2, Selecting from the group consisting of SEQ ID NO:21 and SEQ ID NO:22; and (c) quantifying the amount of ALXN1210 in the biological sample based on the signal ratio of the signature peptide to an internal control. , Wherein the internal control comprises a labeled form of the same signature peptide. The method according to any of the preceding claims, wherein the protease is trypsin. The method of any of the preceding claims, further comprising contacting the biological sample with an affinity capture reagent prior to treating the biological sample with a protease. 23. The method of claim 22, wherein the affinity capture reagent is bead-loaded Protein A. 24. The method of claim 22 or 23, further comprising washing the Protein A binding antibody to remove unbound components prior to proteolysis. The method of any of the preceding claims, further comprising denaturing the antibody sample. The method of any of the preceding claims, further comprising reducing the antibody sample. The method according to any of the preceding claims, further comprising alkylating the antibody sample. The method of any of the preceding claims, wherein the denaturing, reducing, and alkylating unfold the antibody protein and facilitate proteolytic digestion. The method according to any of the preceding claims, wherein the mass spectrometry is reverse phase UPLC-MS/MS. The method according to any of the preceding claims, wherein the biological sample is serum or urine. The method according to any of the preceding claims, wherein the signature peptide comprises no more than 20 amino acids. The method according to any of the preceding claims, wherein the signature peptide comprises no more than 15 amino acids. 20. The method of claim 18 or 19, wherein the signature peptide of eculizumab is SEQ ID NO:19. 20. The method of claim 18 or 19, wherein the signature peptide of eculizumab is SEQ ID NO:20. 21. The method of claim 18 or 20, wherein the signature peptide of ALXN1210 is SEQ ID NO:21. 21. The method of claim 18 or 20, wherein the signature peptide of ALXN1210 is SEQ ID NO:22. The method according to any of the preceding claims, wherein the affinity capture reagent is protein A loaded with magnetic beads. An isolated peptide consisting of SEQ ID NO: 19. An isolated peptide consisting of SEQ ID NO:20. An isolated peptide consisting of SEQ ID NO:21. An isolated peptide consisting of SEQ ID NO:22.

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